GB2178661A - Pharmaceutical compositions containing ubiquinone coenzymes - Google Patents

Abstract

Pharmaceutical compositions having cardiac and muscular metabolic protective activity, containing an ubiquinone coenzyme and creatine phosphate or its salts. Coenzyme Q10 is preferably utilised.

Description

SPECIFICATION Pharmaceutical compositions This invention relates to pharmaceutical compositions having cardiac and metabolic protective activity on muscular energetic metabolisms.

Coenzyme Q10 is known to play a very important role in cellular and tissular respiration processes (Gale P.H. et al.-Arch. Biochem. Biophys.-93-21 1-1961).

Coenzyme Q10 intervenes in the mitochondrial transport of electrons and in oxygen utilization by the cytochrome respiratory chain (Morton R.A.-Nature-182-1764-1959).

Coenzyme Q10 is present in almost all the tissues, particularly in muscles, heart, brain and liver, and evidence for a decreased concentration of Coenzyme Q1O has been found in some pathological conditions of said organs, such as cardiac infarction, myocardiac insufficiency, muscular dystrophy and arterious hypertension, so that one can assume the existence of a relationship between deficiencies in Coenzyine Qic and the above diseases.

Actually, exogenous administration of Coenzyme Q1O proved to restore the impaired tissular concentrations of said coenzyme, and at the same time to display an important therapeutic action (Folkers K. et al.,-lnt. J. Vit. Res.-40-380-1970).

A number of morbid conditions, such as myocardiac insufficiency, post-infarctual conditions, myopathic dystrophies and hypertension were successfully treated by administration of Coenzyme Q1o (Yamasawa.-Biomedical and Clinical Aspects of Coenzyme Q,O-Elsevier-North Holland.-Vol. 1 1-Pag. 333-1980).

Further biochemical and pharmacological investigations have provided evidence that administration of Coenzyme Q1O, besides restoring the depressed mitochondrial respiration, can increase cell ATP concentration and maintain intact the cell membrane.

A similarly important role is that played by creatine phosphate.

A critical concentration of mitochondrial ATP, such as that taking plaace through the cytochrome chain and Coenzyme Q1O, is essential for the production of creatine phosphate, which is synthetized in the mitochondria by mitochrondrial ATP and cytoplasmic creatine.

Said molecule can transport the highly energetic phosphoric bond to myofibrillae and cell membranes.

At this level, in fact, creatine phosphate is used, by means of creatline phosphokinase, to produce ATP from ADP, and to support muscular contraction and ionic transport.

Several experiments have proved that creatline phosphate tissular content is a regulating factor for cardiac contractile strength.

Administration of creatine phosphate prolongs myocardiac contractile function in rat hearts, in reversible ischaemic arrest conditions (Hearse D.J. et al.-J. Pharm. Pharmac.-26-427-1947) and reduces the appearance of ventricular extrasystoles induced by coronary ligation (Marshall R.J., Paratt J.R.-Naunyn-Schmiedebergs' Arch. Pharmacol.-28 1-437-1974).

Moreover, it has been proved that a decrease in high energy level intracellular phosphates leads to impairments of sarcolemma and constitutes the starting of an irreversible ischaemic damage.

Administration of creatine phosphate can prevent depletion of high potential phosphates and inhibit the processes causing the destruction of sarcolemma, as has been shown in tests effected on hearts of ischaemic rats or on the experimental cardiac infarction on the dog, in which test administration of creatline phosphate led not only to a significant improvement in cardiac function and on the levels of high energy phosphates, but also to a higher integrity of myocardiocyte sarcolemma and a concomitant reduction in lysophospholipids produced by degradation of membrane phospholipids (Sako V.A. et al.-J. Molecular and Cellular Cardiology-16, suppl. 2-Sept. 1984; Sharov V.G.-J. Cardiovascular Pharmacol.-1984; Fagliemi O.-J. Cardiovascular Pharm.-4-53-1982).

The results from the tests on the myocardiac protective activity of creatine phosphate were also confirmed in clinical studies in acute cardiac infarction, during which a significant reduction in the appearance of ventricular arrhyhmias was evidenced; in cardiovascular surgery, in which treated patients showed a faster recovery of the hemodynamics and cardiac electric activity and from right ventricle biopsy, where the preservation of normal concentrations of high energy phosphates and a clear protective action on ischaemic injuries of sarcolemma were observed (Robinson L.A., Hearse D.J.-J. of Therapy Cardiovascular Surgery-87-190-1984).

The present invention relates to pharmacueticai compositions comprising as active ingredient a combination of an ubiquinone coenzyme, such as for example Coenzyme Q1O, and the phosphorylated organic compound known as creatine phosphate or N-(phosphonamidino)sarcosine or a salt thereof.

We have found that the components of the combination display a strongly synergetic action, leading to surprising and unexpected results in favouring muscular metabolism and particularly in protecting heart from anoxia-induced injuries which take place for example in cardiac infarction, post-infarction conditions, during cardiovascular surgery or in case of increased muscular energetic requirements.

The surprising pharmacological and therapeutical effects exerted by the pharmaceutical compositions of the present invention are due to the synergism taking place between the ubiquinone component, e.g. Coenzyme Qic, and creatine phosphate.

The synergism was unforeseeable on the grounds of what was hitherto known or from the simple addition of the effects of the single components.

The compositions of the invention may be used in any suitable form, i.e. in solid, semi-solid or liquid form for oral, parenteral or rectal administration. The compositions may be used, for example, in the form of capsules, sugar-coated pills, gastroresistant tablets, ampoules or vials for injectable solution. Any pharmaceutically acceptable excipients may be included. Thus, for example, the active ingredients may be inglobated in lyposomes or lipidic microspheres. Alternatively, the active ingredients may be formulated in ampoules or vials for instant dissolution, containing for example, creatine phosphate sodium salt and optional excipients, in freeze-dried form, in a reservoir plug for instant dissolution in an appropriate Coenzyme Qic solution.

The composition of the invention may be used in human and/or animal therapy for the treatment of pathological conditions related to cardiac and muscular metabolic disorders such as those consequent to myocardiac anoxia, cardiac infarction, cardiovascular surgery muscular fatigability, sport training and generally to conditions of increased muscular energetic requirements.

The results of pharmacological and toxicological tests carried out on the compositions of the invention are described below.

Coenzyme Q1O and creatine phosphate are known to have a very low toxicity, and the combination thereof showed no changes in toxicity. In fact, solutions containing the two components of the combination in ratios from 1:10 to 1:100 (i.e. in greatly higher amounts than those forseeable for therapeutic use) subcutaneously or intraperitoneally injected in the mouse and in the rat, caused no toxic effects. Under these conditions, LD50 proved to be higher than 1 g/kg and 500 mg/kg for subcutaneous and intraperitoneal administration, respectively. The chronic toxicity tests in the rat, carried out orally administrating the above combination in a 1:10 ratio for 30 consecutive days, also showed no pathological changes in body weight and in the different hematochemical parameters.

Protective action against ventricular arryhythmia induced by coronary ligation in the rat The technique described by Selych et al. (Angiology 11-398-1960) and by Clark C. et al. (J.

Pharmacol. Methods 3-357-1980) for the surgical occlusion of the left coronary arteria was applied to male rats of 350-450 g body weight. The rats, which during the surgical operation and before the ligation showed the appearance of arrhythmias or a decrease in arterious pressure under 75 mmHg, were discarded. Coenzyme Qlo and creatine phosphate solutions were slowly injected in the left ventricle lumen, 15 minutes before ligation of coronary arteria.

Arrhythmias started to appear 4-7 minutes after ligation, and ectopic ventricular contractions during 30 minutes after ligation were counted. Coenzyme Qlo and creatine phosphate can reduce the number of ectopic contractions induced by coronary ligation, but this effect is significantly and surprisingiy enhanced by the combination thereof, thus giving evidence of a remarkable synergism, as clearly shown in Table. 1.

TABLE 1

Treatment Start of arrhythmias, Total ectopic contractions (min.) during 30 minutes after ligation Controls 4 - 7 994 + 226 Creatine 4 - 7 870 + 206 phosphate 10 mg/kg Creatine 5 - 7 765 + 215 phosphate 25 mg/kg Coenzyme Q10 5 - 7 709 + 196 5 mg/kg Coenzyme Q10 6 - 7 690 + 222 10 rng/kg Creatine 6 - 7 395 + 176 phosphate 10 mg/kg + Coenzyme Q10 5 rng/kg Creatine 6 - 7 165 + 124 phosphate 25 mg/kg + Coenzyme Q10 10 mg/kg Effect on the ATP content in the papillary muscle of the rabbit after ipoxia A group of New Zealand rabbits of 2 kg average body weight was intravenously treated with a solution containing 10 mg/kg Coenzyme Q10 or 100 mg/kg creatine phosphate, during 3 consecutive days.

Another group of animals was treated with the two drugs combined at the same dosages as above or at one half of the above dosages. All the animals (comprising the controls) were killed 2 hours after the last injection, the hearts were removed and equal sections of the papillary muscle, of 1 mm diameter and 4-5 mm thickness were isolated.

This tissue was analysed in an appropriate thermostatized bath perfused with an 2 100% saturated solution.

The ipoxia conditions were produced by introducing into the bath 100% N2 instead of 02.

The ATP content in the papillary muscle was analyzed according to the procedure described by Strehler B.L. et al. (Methods in Enzymology Ill, New York-Academic Press-871-1957) in tissue samples, both after a balancing period of about 90 minutes in normal perfusion and after 60 minutes of ipoxia.

The obtained results (Table 2) showed that the ATP content was maintained at normal values, even after ipoxia, only in the case of the combination of Coenzyme Qic and creatine phosphate, thus evidencing the synergism between the two compounds.

TABLE 2

Treatment ATP content (mol/g tissue) Before ipoxia After ipoxia Controls 1.73 + 0.29 0.48 + 0.04 Creatine 1.80 + 0.31 0.65 + 0.03 phosphate 100 mg/kg Coenzyme Q10 1.76 + 0.24 0.80 + 0.06 10 mg/kg Creatine 1.88 + 0.27 0.66 + 0.06 phosphate 100 mg/kg + Coenzyme Q10 10 mg/kg Effect on enzymatic activity of gastrocnemius of rats subjected to daily muscular exercise (training) In view of the fact that daily training in the rat leads to an increase of mitochondrial enzyme activity, in both the heart and the gastrocnemius, said increase depending on the duration of training and muscular performances (Benzi G. et al., J. Applied Physiology 38-565-1875) the combination of the invention was tested for any shortening action in the enzymatic adaptation to muscular exercise and therefore of the functional performance.

Injections of creatine phosphate or Coenzyme Q1O proved to increase the mitochondrial enzymatic pool of rat gastrocnemius, but a higher obtainable effect was shown with the combination of the two substances. In this case, the increase in mitochondrial enzymatic activity after 7 days treatment was higher than that obtainable after 30 days training. The animals (Wistar rats) were trained on a Rotarod track (Rotarod apparatus Basile, Comerio-ltaly) and the motorial activity was carried out with 20 m/minutes for 120 minutes every day.

The enzymatic activities were evaulated after 30 days of training, upon isolation and homogenization of the gastrocnemius (Oscai L.B., J. Biol. Med. 246-6968-1971).

The obtained results (Table 3) show that the administration of the combination of the present invention caused an increase of mitochondrial enzymatic activity even higher than that induced by training, said result being not obtained when using the two components separately.

TABLE 3

Treatment Days of Citrate Isocitrate Succinate Training synthetase dehydrogenase dehydrogenase Controls No training 20.8#1.5 2.42#0.19 3.34#0.17 Controls 7 22.2#1.4 2.75#0.16 3.75#0.15 Controls 30 31.7#1.6 3.60#0.22 5.15#0.19 Creatinephosptate No training 22.4#1.2 2.81#0.20 3.80#0.17 100 mg/kg Coenzyme Q10 10 mg/kg No training 26.7#1.9 3.40#0.21 3.84#0.20 Creatine phosphate No training 31.1#1.7 3.10#0.21 4.80#0.17 100 mg/kg + Coenzyme Q10 10 mg/kg Creatine phosphate 7 26.7#1.5 2.96#0.21 3.90#0.19 ( ) 100 mg/kg Coenzyme Q10 7 30.5#1.8 3.35#0.22 4.95 10 mg/kg Creatine phosphate 7 44.1#1.9 5.11#0.20 7.10#0.31 100 mg/kg + Coenzyme Q10 10 mg/kg ( ) Enzymatic activities are expressed as m of used substrate per minute/g of tissue weight.

Claims (9)

1. A pharmaceutical composition, having cardiac and muscular metabolic protecting activities, comprising an ubiquinone coenzyme and creatine phosphate or a salt thereof.

2. A pharmaceutical composition as claimed in claim 1, wherein the ubiquinone coezyme is Coenzyme (tic

3. A pharmaceutical composition as claimed in claim 1 or 2, wherein the coenzyme : creatine phosphate weight ratio is from 1:1000 to 1:2.

4. A pharmaceutical composition as claimed in any one of claims 1 to 3, in solid, semisolid or liquid form, or oral, parenteral or rectal administration, for the treatment of injuries from myocardiac anoxia and muscular metabolic and energetic disorders, in human and/or animal therapy.

5. A pharmaceutical composition as claimed in claim 4, in the form of capsules, sugar-coated pills, gastroresistant tablets, ampoules or vials for injectable solution.

6. A pharmaceutical composition as claimed in any one of claims 1 to 3, wherein the active ingredients are inglobated in lyposomes or lipidic microspheres.

7. A pharmaceutical composition as claimed in any one of claims 1 to 3, wherein the active ingredients are formulated in ampoules or vials for instant dissolution, containing creatine phosphate sodium salt and possible excipients, in freeze-dried form, in a reservoir plug for instant dissolution in an appropriate Coenzyme (tic solution.

8. A pharmaceutical composition according to claim 1 substantially as described herein.

9. For use in the treatment of pathological conditions related to cardiac and muscular metabolic disorders such as those consequent to myocardiac anoxia, cardiac infarction, cardiovascular surgery, muscular fatigability, sport training and generally to conditions of increased muscular energetic requirements, a pharmaceutical composition as claimed in any one of claims 1 to 8.